The Long-term goal of this project is to improve the safety and efficacy of mixed hematopoietic chimerism protocols across major histocompatibility (MHC) barriers such that this approach can be more widely applied for the induction of robust graft tolerance in recipients of islet and solid organ allografts from living and cadaver donors. Stable mixed donor-host hematopoietic chimerism invariably induces robust, lifelong, donor-specific immunologic tolerance to donor tissue grafts. It also restores self-tolerance in autoimmune disorders. Thus this approach is particularly suitable for tolerance induction in islet transplantation. Considerable progress made in reducing the toxicity of the host conditioning regimens required for allogeneic engraftment of hematopoietic cells and in preventing the formidable problem of graft-versus-host disease (GVHD) now provides realistic opportunities for the application of mixed chimerism strategies for tolerance induction in solid organ and cellular transplantation. It is hypothesized that the correct use of anti-CD40L mAb, preferably combined with sirolimus and donor I;ellular antigen, allows, via initial contraction of the alloreactive T cell clone size, induction of regulatory CD4+CD25+ cells, and control of intrathymic alloresistance--the subsequent activation of stable intrathymic mixed hematopoietic chimerism across partial major histocompatibility barriers without the need for T cell depletion, thymic irradiation, and splenectomy. It is also hypothesized that the use of highdose hematopoietic cell transplantation overcomes the need for myeloablation and myelosuppression. To test these hypotheses in the established relevant preclinical model of peripheral blood stem cell transplantation in haploidentical, related nonhuman primates, the following Specific Aims are proposed: SPECIFIC AIM #1: To establish stable mixed hematopoietic chimerism in haploidentical, related nonhuman primate recipients of peripheral blood stem cell transplants under the cover of transient immunosuppression and costimulatory blockade. SPECIFIC AIM #2: To establish that stable mixed hematopoietic chimerism confers tolerance to subsequent same donor islet and skin allografts without compromising the immunocompetence of nonhuman primate recipients of peripheral blood stem cell transplants. SPECIFIC AIM #3: T o use cellular, molecular, and genetic assays to define the underlying mechanisms of action of the approaches used to establish chimerism. The results of these studies will increase our understanding of the safety, efficacy, and underlying mechanisms of selective immunomodulatory approaches aimed at maximizing alloengraftment of haploidentical, related peripheral blood stem cells, thereby providing a basis for the rational design of mixed chimerism strategies in the outpatient setting for the purpose of inducing tolerance to subsequent same living donor islet and/or solid organ allografts.